A MAPK docking site is critical for downregulation of Capicua by Torso and EGFR RTK signaling
2007; Springer Nature; Volume: 26; Issue: 3 Linguagem: Inglês
10.1038/sj.emboj.7601532
ISSN1460-2075
AutoresSergio Astigarraga, Rona Grossman, Julieta Díaz-Delfín, Carmé Caelles, Ze’ev Paroush, Gerardo Jiménez,
Tópico(s)Plant Molecular Biology Research
ResumoArticle25 January 2007free access A MAPK docking site is critical for downregulation of Capicua by Torso and EGFR RTK signaling Sergio Astigarraga Sergio Astigarraga Institut de Biologia Molecular de Barcelona-CSIC, Parc Científic de Barcelona, Barcelona, Spain Search for more papers by this author Rona Grossman Rona Grossman Department of Biochemistry, Faculty of Medicine, The Hebrew University, Jerusalem, Israel Search for more papers by this author Julieta Díaz-Delfín Julieta Díaz-Delfín Institut de Recerca Biomèdica, Parc Científic de Barcelona, Barcelona, Spain Search for more papers by this author Carme Caelles Carme Caelles Institut de Recerca Biomèdica, Parc Científic de Barcelona, Barcelona, Spain Search for more papers by this author Ze'ev Paroush Ze'ev Paroush Department of Biochemistry, Faculty of Medicine, The Hebrew University, Jerusalem, Israel Search for more papers by this author Gerardo Jiménez Corresponding Author Gerardo Jiménez Institut de Biologia Molecular de Barcelona-CSIC, Parc Científic de Barcelona, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Search for more papers by this author Sergio Astigarraga Sergio Astigarraga Institut de Biologia Molecular de Barcelona-CSIC, Parc Científic de Barcelona, Barcelona, Spain Search for more papers by this author Rona Grossman Rona Grossman Department of Biochemistry, Faculty of Medicine, The Hebrew University, Jerusalem, Israel Search for more papers by this author Julieta Díaz-Delfín Julieta Díaz-Delfín Institut de Recerca Biomèdica, Parc Científic de Barcelona, Barcelona, Spain Search for more papers by this author Carme Caelles Carme Caelles Institut de Recerca Biomèdica, Parc Científic de Barcelona, Barcelona, Spain Search for more papers by this author Ze'ev Paroush Ze'ev Paroush Department of Biochemistry, Faculty of Medicine, The Hebrew University, Jerusalem, Israel Search for more papers by this author Gerardo Jiménez Corresponding Author Gerardo Jiménez Institut de Biologia Molecular de Barcelona-CSIC, Parc Científic de Barcelona, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Search for more papers by this author Author Information Sergio Astigarraga1, Rona Grossman2, Julieta Díaz-Delfín3, Carme Caelles3, Ze'ev Paroush2 and Gerardo Jiménez 1,4 1Institut de Biologia Molecular de Barcelona-CSIC, Parc Científic de Barcelona, Barcelona, Spain 2Department of Biochemistry, Faculty of Medicine, The Hebrew University, Jerusalem, Israel 3Institut de Recerca Biomèdica, Parc Científic de Barcelona, Barcelona, Spain 4Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain *Corresponding author. Department of Molecular and Cellular Biology, Institut de Biologia Molecular de Barcelona-CSIC, Parc Cientific de Barcelona, Josep Samitier, 1-5, Barcelona 08028, Spain. Tel.: +34 934 034 970; Fax: +34 934 034 979; E-mail: [email protected] The EMBO Journal (2007)26:668-677https://doi.org/10.1038/sj.emboj.7601532 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Early Drosophila development requires two receptor tyrosine kinase (RTK) pathways: the Torso and the Epidermal growth factor receptor (EGFR) pathways, which regulate terminal and dorsal-ventral patterning, respectively. Previous studies have shown that these pathways, either directly or indirectly, lead to post-transcriptional downregulation of the Capicua repressor in the early embryo and in the ovary. Here, we show that both regulatory effects are direct and depend on a MAPK docking site in Capicua that physically interacts with the MAPK Rolled. Capicua derivatives lacking this docking site cause dominant phenotypes similar to those resulting from loss of Torso and EGFR activities. Such phenotypes arise from inappropriate repression of genes normally expressed in response to Torso and EGFR signaling. Our results are consistent with a model whereby Capicua is the main nuclear effector of the Torso pathway, but only one of different effectors responding to EGFR signaling. Finally, we describe differences in the modes of Capicua downregulation by Torso and EGFR signaling, raising the possibility that such differences contribute to the tissue specificity of both signals. Introduction Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that translate extracellular signals into changes in gene expression and cellular function. Many RTKs signal through the evolutionarily conserved Ras/MAPK cassette, in which a cascade of interacting kinases ultimately leads to phosphorylation of nuclear transcription factors. As essentially the same Ras/MAPK cassette is used in multiple developmental contexts, a fundamental question has been how Ras/MAPK signals are interpreted to produce tissue-specific outputs (Rommel and Hafen, 1998; Tan and Kim, 1999). Here, we investigate the responses of a single nuclear protein, Capicua, to two distinct RTK pathways during Drosophila development. Patterning of the Drosophila embryo and its surrounding eggshell requires two different RTK pathways initiated by the transmembrane receptors Torso and Epidermal growth factor receptor (EGFR). The Torso pathway controls the specification of the most anterior and posterior (terminal) regions of the embryo (Duffy and Perrimon, 1994; Furriols and Casanova, 2003). An extracellular signal produced during oogenesis activates Torso at each pole of the blastoderm embryo, leading to stimulation of the Ras/MAPK pathway, and hence, to localized activation of the gap genes tailless (tll) and huckebein (hkb) in terminal positions (Pignoni et al, 1990; Brönner and Jäckle, 1991; Duffy and Perrimon, 1994; Furriols and Casanova, 2003). Although regulation of tll and hkb at the anterior pole is complex and requires additional inputs from the anterior and dorsal-ventral (DV) maternal systems (Pignoni et al, 1992; Brönner et al, 1994; Reuter and Leptin, 1994), the activation of tll and hkb at the posterior pole occurs mainly in response to the Torso pathway. This activation, however, is indirect and involves a mechanism of derepression: both genes are normally repressed in central regions of the embryo and the Torso signal counteracts this repression to permit localized tll and hkb transcription at the pole (Liaw et al, 1995; Paroush et al, 1997; Jiménez et al, 2000). Repression of tll and hkb requires several nuclear factors such as the high-mobility group (HMG) protein Capicua (Cic) and the Groucho (Gro) corepressor. Embryos lacking maternally contributed Cic or Gro show derepression of tll and hkb towards the center of the embryo, which then leads to suppression of the thoracic and abdominal primordia (Paroush et al, 1997; Jiménez et al, 2000). Cic has been proposed to be the target of the Torso inhibitory signal because the protein appears excluded from the embryo poles in a Torso-dependent manner (Jiménez et al, 2000). However, it is not known whether this exclusion is direct, nor whether it is essential for the interpretation of the Torso signal. Earlier in development, EGFR signaling also appears to establish antagonistic interactions with Cic. The EGFR pathway patterns the DV axis of both the embryo and the eggshell (Ray and Schüpbach, 1996). During mid-oogenesis (stage 9), the Gurken ligand is secreted at the dorsal-anterior corner of the oocyte and activates the EGFR present in the adjacent somatic follicle cells, thus conferring to these cells a dorsal fate. Subsequently, the dorsal-anterior follicle cells secrete the dorsal respiratory appendages of the eggshell, whereas the ventral follicle cells produce an extracellular signal towards the oocyte that will specify the ventral regions of the embryo. Mutations that prevent EGFR signaling lead to ventralization of the eggshell and the embryo, whereas ectopic EGFR activation dorsalizes the egg (Schüpbach, 1987; Queenan et al, 1997). Dorsalization phenotypes are also produced by several cic loss-of-function alleles, indicating that Cic normally prevents ventral and lateral follicle cells from acquiring a dorsal fate (Goff et al, 2001; Atkey et al, 2006). Additionally, Cic has been reported to accumulate in the nucleus of follicle cells except in dorsal-anterior regions, where EGFR is active (Goff et al, 2001). Again, the functional importance of this exclusion remains untested. In this paper, we show that Cic is a direct nuclear target of the Torso and EGFR pathways. We identify a critical regulatory motif in Cic (designated C2) that functions as a MAPK docking site and interacts with Rolled, the MAPK acting in the Torso and EGFR pathways (Biggs and Zipursky, 1992; Biggs et al, 1994; Brunner et al, 1994). Mutations in the C2 motif generate Cic derivatives that escape downregulation by Torso and EGFR signaling and produce phenotypes that resemble those resulting from the loss of Torso and EGFR function. These results indicate that Cic downregulation is essential for both terminal and DV patterning. We also describe differences in the modes of Cic downregulation by Torso and EGFR signaling and discuss the possible mechanisms underlying those Cic responses. Results Independent motifs in Cic mediate transcriptional repression and downregulation by Torso We first sought to identify the domains required for Cic repressor activity and for downregulation by Torso. Cic orthologs from invertebrate and vertebrate species share two well-conserved regions: the HMG-box presumed to mediate binding to target promoters and a C-terminal domain of unknown function (Jiménez et al, 2000; Figure 1A). To test if these two regions might suffice to provide Cic function, we designed a mini-Cic derivative (Cicmini) containing both regions, but lacking most of the remaining non-conserved sequences (Figure 1B). The Cicmini construct was tagged with the hemaglutinin (HA) epitope, placed under the control of cic genomic sequences and transformed into flies (Materials and methods). Maternal expression of Cicmini rescues the phenotypes caused by the cic1 mutation (Figure 1C–E; Jiménez et al, 2000). This indicates that Cicmini is able to repress tll and hkb in the central regions of the embryo. Furthermore, Cicmini accumulates efficiently in the embryo, but is clearly absent from the poles (Figure 1G and H), suggesting that it also responds to Torso-mediated downregulation. Thus, the critical domains mediating Cic activity and regulation are contained within the Cicmini construct. Figure 1.Different motifs in Cic mediate repressor function and downregulation by Torso. (A) Diagram of Cic protein and evolutionary conservation of the C1 and C2 motifs. The Drosophila sequences shown correspond to residues 1345–1355 (C1) and 1052–1071 (C2); the total length of Cic is 1403 amino acids. Identical and similar residues are boxed in black and gray, respectively. Ag, Anopheles gambiae; Am, Apis mellifera; Ce, Caenorhabditis elegans; Hs, Homo sapiens; Mm, Mus musculus; Tn, Tetraodon nigroviridis. The distantly related C2-like sequences from vertebrate orthologs are present in approximately equivalent positions between the HMG box and the C-terminus of those proteins. (B) Diagram of Cic derivatives expressed under the control of cic regulatory sequences. The HA tag is represented by a gray oval. The rescue activities and regulation of each construct are indicated on the right. The rescue activity of CicΔC2 was assayed using weak cicΔC2 insertions that do not cause embryonic lethality in one copy (asterisk). (C) Embryonic cuticle of wild-type embryo. (D) cic1 mutant embryo with strongly suppressed trunk and abdomen. (E) cic1 mutant embryo rescued by maternal expression of Cicmini. (F) cic1 embryo showing only partial rescue by CicΔC1. (G–L) Distribution of different Cic derivatives at the posterior of blastoderm embryos. Embryos were stained with anti-HA antibody (green) and rhodamine–phalloidin (red) to visualize the cortical actin associated with the plasma membrane. Wild-type Cic (G), Cicmini (H), CicminiNLS (J) and CicΔC1 (K) proteins exhibit significant downregulation at the pole. In contrast, CicΔC2 accumulates ectopically at the pole and in germ cells (L). Note the cytoplasmic localization of CicΔHMG and its accumulation at the pole (I). Identical results were obtained at the anterior of the embryo (Supplementary Figure 1 and data not shown). Download figure Download PowerPoint Next, we assayed the effects of mutating the conserved domains of Cic. We first tested a Cic derivative lacking the HMG domain (CicΔHMG; Figure 1B). Expression of CicΔHMG fails to rescue cic1 embryos (data not shown), demonstrating the requirement of the HMG domain for Cic function. Moreover, CicΔHMG localizes predominantly in the cytoplasm of blastoderm embryos (Figure 1I), suggesting that the HMG region contains nuclear localization signals (NLSs) that target Cic to the nucleus. This cytoplasmic localization of CicΔHMG is not the sole cause underlying its lack of rescuing activity, as an additional derivative (designated CicminiNLS; Figure 1B), in which the HMG domain of Cicmini was replaced by the NLS from SV40 T antigen (Kalderon et al, 1984), is unable to rescue the cic1 mutant phenotype despite being nuclear (Figure 1B and J; data not shown). Thus, the HMG domain has additional roles other than mediating nuclear localization, most probably the binding to regulatory DNA sequences in target genes. We also find that CicΔHMG is clearly detectable at the embryonic poles, suggesting that it is insensitive to downregulation by Torso signaling (Figure 1I). This could indicate that Cic downregulation requires access of the protein to the nucleus, or that the HMG domain is directly involved in the mechanism of Cic downregulation. In support of the first possibility, the nuclear CicminiNLS derivative displays significant downregulation at the poles in the absence of a functional HMG domain (Figure 1J). We distinguish two conserved motifs within the C-terminal region of Cic, which we designate as C1 and C2. C1 is a 48-amino-acid sequence containing a highly invariable core of 11 residues present in all Cic orthologs (Figure 1A; Jiménez et al, 2000). We find that a C1-deleted derivative (CicΔC1; Figure 1B) produces only partial rescue of cic1 mutant embryos (Figure 1F). This suggests that the C1 motif is important for Cic repressor activity. In contrast, this motif is dispensable for Torso-dependent regulation because CicΔC1 is clearly suppressed at the embryo poles (Figure 1K). The C2 motif is highly conserved in Cic orthologs from insects and nematodes, but appears to have diverged in vertebrate Cic proteins (Figure 1A). We find that the expression of a Cic derivative carrying a deletion of C2 (CicΔC2; Figure 1B) has a strong maternal effect, resulting in embryonic lethality. The penetrance of this effect is >95% with a single copy of the transgene in approximately half of the transgenic lines (data not shown). The remaining lines cause embryonic lethality only when two copies are present. A single copy of weak cicΔC2 insertions rescues the cic1 mutation to adulthood (data not shown), indicating that CicΔC2 is a functional repressor. Remarkably, CicΔC2 accumulates extensively at the embryo poles and in pole cell nuclei (Figure 1L and Supplementary Figure 1). This ectopic accumulation of CicΔC2 is also observed in cic1 mutant embryos that lack endogenous Cic protein (data not shown), suggesting that this result does not simply reflect saturation of the downregulation machinery by the combined expression of CicΔC2 and endogenous Cic. Thus, the C2 motif is specifically required for Cic downregulation in response to Torso signaling. Embryos expressing strong cicΔC2 insertions frequently lack the eighth abdominal segment and the posterior spiracle (Figure 2A), a phenotype shared by mutants lacking Torso activity (Duffy and Perrimon, 1994). Stronger defects extending to the abdominal region are also observed in approximately 40% of the embryos (Figure 2B). We hypothesized that CicΔC2 is a dominant repressor that escapes downregulation by Torso and consequently perturbs posterior terminal development. Indeed, cicΔC2 embryos exhibit reduced or absent posterior expression of tll and hkb (Figure 2C–F), indicating that CicΔC2 represses both genes even in terminal nuclei exposed to Torso activity. We also examined the expression of hunchback (hb), which normally forms a posterior stripe under the control of tll and hkb function (Casanova, 1990; Brönner and Jäckle, 1991; Figure 2G). As shown in Figure 2H, this hb stripe is shifted towards the pole in cicΔC2 embryos, again indicating that CicΔC2 interferes with posterior terminal patterning. In contrast, the effects of CicΔC2 are much weaker at the anterior pole, resulting in reduced hkb expression and mildly affected patterns of tll and hb (Figure 2C–H; see Discussion). We conclude that Cic downregulation is essential for the correct specification of posterior terminal structures and critically depends on the C2 motif. Figure 2.CicΔC2 interferes with embryonic terminal development. (A, B) Cuticle phenotypes resulting from maternal expression of CicΔC2. Shown are examples of intermediate phenotypes characterized by the lack of posterior terminal structures including the A8 segment (A, arrowhead), and stronger effects where additional segments (typically A6 and A7, arrowheads) are affected (B). (C–H) mRNA expression patterns of tll (C, D), hkb (E, F) and hb (G, H) in wild-type (C, E, G) and cicΔC2 (D, F, H) embryos. The mutant embryos exhibit marked repression of tll and hkb at the posterior pole, and shifted expression of the posterior hb stripe. At the anterior region, hkb expression appears markedly reduced (arrowhead in F). We also note a slight anterior shift (of approximately 6% egg length) of the dorsal tll stripe (arrowhead in D). Download figure Download PowerPoint The C2 motif is a MAPK docking site The C2 element contains a TP dipeptide (amino acids 1059–1060 in Cic; Figure 1A) that could be a potential MAPK phosphorylation site in response to Torso signaling. To test if phosphorylation of T1059 mediates downregulation of Cic, we generated Cic derivatives in which T1059 was replaced by either A or D residues that should prevent or mimic phosphorylation of this site, respectively. If phosphorylation of T1059 controls Cic downregulation, the T1059A mutation should be refractory and therefore more disruptive to this downregulation than T1059D. We, however, find that T1059D causes stronger suppression of Cic regulation than T1059A, suggesting that phosphorylation of T1059 is unlikely to explain the function of the C2 motif (Supplementary Figure 2). We then tested the possibility that the C2 motif functions as a MAPK docking site for Rolled (Tanoue and Nishida, 2003). Using the yeast two-hybrid system we found that, indeed, a Cic fragment comprising the C1 and C2 motifs interacts strongly with Rolled (data not shown). Further experiments showed that the binding maps precisely to the C2 element (Figure 3A and B). The binding requires the C-terminus of Rolled (Figure 3B), which spans the common docking (CD) domain mediating association of MAPKs to their substrates (Tanoue et al, 2000). The C2 motif also interacts with the human Erk2 MAPK (Figure 3B). We independently confirmed the association between the C2 motif and Rolled using in vitro pull-down assays. A Cic fragment containing the C2 motif interacts with Rolled, whereas the equivalent fragment lacking the C2 core does not (Figure 3A and C; see also Supplementary Figure 2F). Furthermore, the C2 element mediates phosphorylation of Cic by Erk2 in vitro: activated Erk2 immunopurified from HeLa cells efficiently phosphorylates a Cic fragment including the C2 motif, but not the equivalent C2-deleted fragment (Figure 3A and D). This phosphorylation is Erk2-specific because it is not observed while using activated Jun kinase (JNK; Figure 3D). Collectively, our results indicate that C2 is a docking motif that recruits Rolled to induce phosphorylation of Cic in response to signaling. Like other MAPK docking sites, the C2 element includes several conserved hydrophobic residues (Tanoue and Nishida, 2003), although their specific arrangement is different, suggesting that C2 represents a novel MAPK docking motif. Figure 3.The C2 motif is a MAPK docking site. (A) Diagram of Cic protein showing the different fragments assayed for binding in yeast and in vitro. The limits of each Cic fragment are as follows: Cic1 (1050–1115), Cic2 (1307–1379), Cic3 (1050–1079) and Cic4 (942–1116). The C2 amino-acid sequences deleted in Cic1ΔC2 and Cic4ΔC2 are 1054–1064 and 1052–1072, respectively. (B) Yeast two-hybrid assay using different LexA-Cic baits and B42 fusion preys. Positive interactions (visualized by lacZ reporter activation) are observed between C2-containing fragments and Rolled or human Erk2. Negative controls are RolledΔCD, a form of Rolled lacking the C-terminal 52 amino acids that should be unable to bind substrates, and Hairy. (C) Pull-down assay using the indicated GST fusions and in vitro translated (IVT) Rolled or Luciferase (Luc) labeled with 35S-methionine. Cic4 binds Rolled in a C2-dependent manner. Negative controls show little or no interaction between Hairy and Rolled, or between Cic and Luciferase. Input lanes were loaded with 10% of the protein used in the binding reactions. (D) In vitro phosphorylation assay using the indicated GST fusions and either Erk2 or Jun kinase (JNK) protein immunopurified from HeLa cells unstimulated (−) or stimulated (+) for kinase activation. Positive control substrates for Erk2 and JNK were myelin basic protein (MBP) and Jun, respectively. Erk2 specifically phosphorylates Cic4 but not Cic4ΔC2. The gels shown in (C) and (D) were stained with Coomassie to ensure equal loading and integrity of all GST fusions (not shown). Download figure Download PowerPoint EGFR signaling alters the subcellular distribution of Cic in the ovary We next investigated the regulation of Cic by EGFR signaling in the ovary. First, we re-examined the distribution of Cic in follicle cells using the sensitive HA-tagged version of the protein, which is able to rescue the fettucine (fet) alleles of cic that cause dorsalization of the egg (data not shown; Goff et al, 2001). In these experiments, we also monitored expression of mirror, an EGFR-induced target that marks the dorsal-anterior region of the egg chamber (Jordan et al, 2000; Zhao et al, 2000). During stage 9–10A, Cic accumulates in the nuclei of follicle cells located outside the dorsal-anterior region (Figure 4A–D). Unexpectedly, we also detect Cic in dorsal-anterior cells, albeit equally distributed between the nucleus and the cytoplasm (Figure 4A–D; see also Supplementary Figure 3). This localization of epitope-tagged Cic was also observed in a cicfetU6/cicfetE11 mutant background with reduced levels of endogenous Cic (data not shown), suggesting that accumulation of Cic in dorsal-anterior cells occurs at physiological levels of Cic expression. Later in oogenesis (stages 11–12), Cic regulation appears confined to a small patch of only ∼40 dorsal follicle cells comprised within a broader area of Mirr expression (Figure 4E and F), suggesting that the domain of Cic downregulation has retracted towards the dorsal midline, whereas the Mirr expression domain has remained unaltered. Also, the late patch is divided by a stripe of cells in the presumptive midline in which Cic is preferentially nuclear (Figure 4F; empty arrowhead). This stripe coincides with the midline region in which EGFR signaling declines at this stage (Wasserman and Freeman, 1998; Peri et al, 1999; also see below). By stages 12–13, the patch of Cic regulation includes only 15–20 cells and the midline stripe of nuclear protein is still visible (data not shown). Thus, Cic regulation in follicle cells is dynamic and correlates with the evolving pattern of EGFR activity. Figure 4.EGFR signaling induces nucleocytoplasmic redistribution of Cic via the C2 motif. (A) Partial view of a stage-10 cic-HA egg chamber costained with anti-HA (green, A) and anti-Mirror (Mirr; blue, A′) antibodies, and with rhodamine–phalloidin (red, A′) to label the cortical actin. The merged image is shown in (A″). (B) Detail of the dorsal-anterior region of the above egg chamber; expression of Mirr is not shown. The merged image is shown in (B′). The preferential nuclear accumulation of Cic in some cells (asterisk in B) probably results from regional differences in the levels of Ras/MAPK activation within the dorsal-anterior patch at this stage (Peri et al, 1999). (C, D) High magnifications of two areas from (B′) corresponding to dorsal-anterior (C, 1) and lateral (D, 2) follicle cells. Cic accumulates in the cytoplasm of dorsal-anterior but not lateral follicle cells (arrows). The central areas of nuclei devoid of staining probably correspond to nucleoli (empty arrowheads). (E) Dorsal view of a stage-11 cic-HA egg chamber stained with anti-HA (green, E) and anti-Mirr (red, E′) antibodies. The merged image is shown in (E″). Note the nuclear accumulation of Cic in cells that express Mirr (arrowheads) and the preferential nuclear accumulation of Cic in a row of cells in the presumptive midline (open arrowhead). (F) Detail of the dorsal-anterior region of the egg chamber shown in (E). (G) Partial view of a stage-10 cicΔHMG-HA egg chamber stained as in (E). (H) Detail of the boxed area shown in (G). CicΔHMG is excluded from the dorsal-anterior nuclei (arrow), whereas only the nucleoli remain devoid of protein in more posterior cells (open arrowhead). (I) Partial view of a stage-10 cicΔC2-HA egg chamber stained as in (E). In the merge panels, colocalization of the green and red channels appears in yellow–orange, whereas colocalization of green and blue channels appears in cyan. Stages of egg chambers are indicated. nc, nurse cells. Download figure Download PowerPoint We examined the localization of our Cic mutant derivatives in the ovary. Specifically, we find that CicΔHMG accumulates in both the nucleus and cytoplasm of cells located outside the dorsal-anterior patch, and is exclusively cytoplasmic in this patch (Figure 4G and H). Moreover, the amount of cytoplasmic CicΔHMG protein appears elevated in dorsal-anterior cells, suggesting that CicΔHMG becomes concentrated in the cytoplasm upon EGFR signaling and the total amount of protein in the cell remains unaltered. This result further indicates that Cic regulation in the ovary occurs at the level of nucleocytoplasmic localization rather than through degradation. In contrast, CicΔC2 is exclusively nuclear in all follicle cells, including those at dorsal-anterior regions (Figure 4I). This indicates that redistribution of Cic in response to EGFR signaling depends on the C2 motif. Notably, nuclear CicΔC2 does not affect Mirr expression (Figure 4I″), suggesting that EGFR-induced activation of mirr is not just a consequence of Cic downregulation (see Discussion). DV patterning defects induced by CicΔC2 Unregulated accumulation of CicΔC2 in the dorsal-anterior follicle cells causes DV patterning defects. Females expressing strong cicΔC2 combinations produce eggs with partial or complete fusions of their dorsal respiratory appendages (Figure 5A–C). These ventralization phenotypes resemble those arising from insufficient EGFR activity in dorsal-anterior follicle cells (Schüpbach, 1987), indicating that EGFR-mediated downregulation of Cic is essential for the correct patterning of the eggshell. Figure 5.CicΔC2 causes DV patterning defects in the egg. (A) Dorsal-anterior region of a wild-type eggshell showing the two symmetrical respiratory appendages. (B, C) Equivalent views of cicΔC2 eggshells showing moderate (B) and severe (C) fusions of appendages. The frequencies of phenotypic classes resulting from strong cicΔC2 combinations are as follows: wild-type, 40%; moderate, 25%; severe, 30%. (D–G) mRNA expression patterns of twi (D, E) and zen (F, G) in wild-type (D, F) and cicΔC2 (E, G) embryos. Download figure Download PowerPoint Although cicΔC2 embryos do not show marked signs of ventralization in their cuticles, they do display altered expression of early ventral and dorsal markers such as twist (twi) and zerknüllt (zen) (Rusch and Levine, 1996). In wild-type embryos, twi forms a band of expression on the ventral side (Figure 5D). In comparison, more than 70% of cicΔC2 embryos show expanded twi expression towards the dorsal side, particularly in posterior regions (Figure 5E). Additionally, the dorsal-specific expression of zen retracts in cicΔC2 embryos, being almost absent in dorsal-posterior positions (Figure 5F and G). Thus, cicΔC2 embryos are moderately ventralized, raising the possibility that downregulation of Cic by EGFR signaling during oogenesis is important for establishing the DV pattern of the embryo. It should be noted, however, that the reduced zen expression at or near the posterior pole of cicΔC2 embryos could also be a consequence of ectopic CicΔC2 repressor activity at this position, as we have previously proposed that Cic participates in dorsal-mediated repression of zygotic targets such as zen (Jiménez et al, 2000). To further examine how CicΔC2 interferes with embryonic DV patterning, we determined its effects on pipe expression in the ovary. pipe encodes a sulfotransferase expressed in ventral follicle cells that initiates the specification of the embryonic DV pattern (Sen et al, 1998; Figure 6A). We find that most cicΔC2 egg chambers sho
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